Membrane fusion is required for neurotransmitter release at the nerve terminal. Formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex bridges the neurotransmitter-loaded synaptic vesicles to the presynaptic plasma membrane, facilitating membrane fusion. The long-term goal of this project is to elucidate the molecular mechanism of SNARE assembly and SNARE-induced membrane fusion. SNARE proteins are amphipathic integral membrane proteins that are not currently amenable to x-ray crystallography and NMR. The present project uses site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), an established technique for the investigation of structures and topologies of membrane proteins. On the basis of various EPR results a structural model of the protein in the native-like phospholipid bilayer is generated at backbone resolution. In this project, structures and membrane topologies of individual full-length SNARE proteins, their assembly intermediates, and the final complex are determined using SDSL EPR. In particular, emphasis is placed on the domains at the membrane-water interface and the transmembrane domains that are directly involved in driving lipid mixing and fusion pore formation. Amino acid sequences of neuronal SNAREs are similar to those of other SNAREs involved in the endocytic and secretory pathways. Further, the structure of the soluble SNARE core is astonishingly similar to those of viral fusion proteins such as influenza hemagglutinin and HIV gp41. Therefore, what is learned from neuronal SNAREs will have strong implications for other membrane fusion systems. Dysfunction of SNARE assembly results in severe mental illness. Thus, this study will help understand the molecular mechanism of SNARE-related mental illness.